Cardiac gap junctions electrically couple adjacent myocardial cells to mediate action potential propagation. These membrane channels not only regulate normal conduction that coordinates the beat-to-beat contraction of the heart but also arrhythmias such as ventricular tachycardia and fibrillation, responsible for about 250, 000 deaths each year. Electron cryo-microscopy and image analysis of two-dimensional crystals is a powerful approach to study the structure of biological membranes because the proteins are maintained in a lipid bilayer environment in physiological buffers. In the last funding cycle, analysis of double membrane gap junction crystals enabled us for the first time to derive a 3D map at 7.5 Angstroms in-plane and 20 Angstroms vertical resolution, which showed that the two hexameric connexons in the dodecameric channel are staggered by 30 degrees with respect to each other. Each connexon is formed by 24 closely packed alpha-helices, and each connexin subunit contains 4 transmembrane alpha-helices, two exposed to the lumen of the pore and two exposed to the membrane lipids. The extracellular protein density forms a tight seal to prevent the exchange of ions and metabolites with the extracellular space. On the basis of (1) interpretation of a preliminary map at 5.7 Angstroms in-plane resolution, (2) energy calculations for over 3 million different alpha-helical models, (3) analysis of multiple connexin sequences, and (4) published results on chemical modification, site-specific mutagenesis and electrophysiology, we have proposed a working model in which helices M3 and M1 are the major and minor pore-lining helices, respectively, and helices M2 and M4 are exposed to the membrane lipids. Threads of density in the extracellular region suggest that beta sheets may stabilize the end-to-end docking of the two connexons. A major goal for the next funding cycle will be to increase the resolution of the map so that we can test the validity of our hypothetical model. In addition, we will continue to investigate molecular mechanisms for channel gating. A comparison of channels in the presence and absence of oleamide will examine how this lipid like molecule blocks intercellular communication. Analysis of full-length channels will test whether the carboxy-tail is a "ball-and-chain" that mediates channel closure at acid pH. Due to the limited vertical resolution of our 3D maps, as well as the exceedingly low yield of crystals, continued progress necessitates that effort be devoted to molecular modeling, as well as technical issues such as protein expression and automated methods to accelerate data collection. This work will be of general utility for membrane protein structure analysis by electron cryo-crystallography. Thus far our studies have revealed the basic structural design of gap junction channels. High resolution structures in the open and closed states would provide considerable insight into the molecular basis of current flow in the heart and will be essential to guide the design of novel agents to treat arrhythmias.